CYTOKINES FROM NATURAL KILLER OR T CELLS

Abstract: Cell-free preparations of a cytokine produced by natural killer (NK) cells activated with interleukin 2 (IL-2) or produced by CD4<+> T cells activated with anti-CD3 monoclonal antibody are described. This cytokine induces antibody secretion by activated B cells. Methods for producing the cell-free supernatants containing the cytokine are also described.

Description: CYTOKINES FROM NATURAL KILLER OR T CELLS I. GOVERNMENT INTEREST This invention described herein may be manufactured, licensed and used by or for the United States Government without the payment of any royalties to us thereon. The Federal Government has a nonexclusive nontransferable, irrevocable, paid-up license to practice or have practiced for or on behalf of the United States any subject invention throughout the world. II. TECHNICAL FIELD OF THE INVENTION The invention is in the field of cytokines. More specifically it is in the field of cytokines which potently induce antibody secretion by activated animal B cells. III. BACKGROUND OF THE INVENTION Cytokines are proteinaceous substances or protein factors whose activity is derived from T cells or Natural Killer (NK) cells. They serve as the biological center piece or source of origin for a variety of biological activities because of their ability to potently induce antibody secretion by activated animal B cells. As a natural consequence of this ability, derivative products can be made from cytokines which further expand the scope of their enormous utility in the medical and research community. The specificity of the cytokines has raised great hopes for their use and products derived therefrom in the prevention and therapy of abnormal health conditions. They can be used individually or in combination with other cytokines to stimulate antibody production in order to modulate complex responses such as immune responses. Thus, there is a continuing need and interest in the medical arts for new members of the cytokine arsenal useful in the battle to prevent, treat and/or ameliorate the deleterious effects of infection and disease states which affect the world community. IV. SUMMARY OF THE INVENTION It is an object of this invention to provide a novel cytokine. In accordance with this invention, and as broadly described herein, a second object of this invention is to provide a cytokine which can potently induce antibody secretion by activated animal B cells. These and other embodiments of the invention are fulfilled by the following embodiments, as illustrated in a general discussion of cytokines derived from T cells and NK cells. In one embodiment a cell-free preparation of a novel active substance whose activity is derived from T cells or NK cells is provided which potently induces antibody secretion by activated animal B cells; resists neutralization by monoclonal antibodies against cytokines produced by NK cells activated by IL-2 or neutralization by monoclonal antibodies or receptor antagonists against cytokines produced by activated T cells; has Ig secretion activity which is not mimicked by the addition of known T cell cytokines or NK cell cytokines when added to separate cell-free preparations in the absence of applicants' novel cell-free preparations of cytokines. Additionally, relative to the active substance whose activity is derived from T cells, it has been discovered that it is a protein; can be produced by activation of the T cell through the T cell receptor signalling pathway which induces cytokine production by T cells; has -activity which is inhibited by treatment with a proteolytic enzyme; has activity which is optimal within 3 to 5 hours after activation of the T cell; has detectable activity at dilution levels as low as 1:3000 and operates as a late acting factor required to induce proliferated B cells to mature into antibody secreting cells. It appears that the similarity between the cytokines discovered by applicants as being derived from NK cells or T cells range from substantially the same to identical. In another embodiment of the invention a method of producing a cytokine preparation is provided, comprising: coating the bottom of a flask with anti-CD3 antibody; adding T cells into the flask and allow the T cells to remain in the flask for a period of 24 hours; harvesting the cell-free supernatant; inducing the activation and proliferation of B cells in the cell- free supernatant through the B cell antigen receptor mediated signalling pathway utilizing dextran conjugated anti-IgD antibodies in the presence of Interleukin-1 and Interleukin-2 wherein the B cells are at least 98.5 percent purified through fluorescence- activated cell sorting (FACS) ; adding activated T cell supernatant; harvesting the B cell culture supernatant 6 days after culture; and measuring the concentration of secreted IgM in the B cell cultured supernatant by an enzyme-linked Immunosorbent Assay (ELISA) . These and other embodiments will be apparent to those skilled in the art from the detailed disclosure which follows. Cytokines prepared in accordance with this invention are derived from mammalian cells, including humans, and can be used generally to stimulate the immune system in immunocompromised hosts. Their utility extends to such areas as adjuvants for boostering mammalian immune responses to vaccination, organ transplantation, chemotherapy, radiotherapy, genetic immunodeficiencies, and AIDS (autoimmune deficiency syndrome) or ARC (AIDS related complex. Additionally, an antagonist or monoclonal antibody to neutralize the cytokine can be made or uniquely identifying and isolating its receptor on the surface of the B cell would be useful in the modulation of antibody production. Moreover, the identification of the receptor on the surface of the B cell can be utilized to create an antagonist (soluble receptor to prevent binding of the cytokine to the receptor. Further, cytokines may be used alone or in combination with other cytokines (i.e., as pharmaceutical cocktail) to stimulate antibody production. V. BRIEF DESCRIPTION OF THE FTGURFS Figure 1 depicts total, RBC-lysed, spleen cells that were stained with FITC-labelled anti-CD3 mAb (2C11) and phycoerythrin-labelled polyclonal goat anti-mouse igM (left upper panel [unsorted]). B cells are IgM + CD3-, T cells are IgM"CD3 + , and non-B, non-T cells are IgM"CD3-. Re-analyses of sorted cells are displayed in right upper panel (B cells) , left lower panel (T cells) , and right lower panel (non-B, non-T cells) . Figure 2 shows B « and B* cells that were cultured at 1.25 x 10* cells/ml with αS-dex (3 ng/ml) and/or IL-2 (150 U/ml) in the absence or presence of ιo% non-B, non-T cells (IgM-CD3 " ) . six days after initiation of culture, SN was harvested for determination of secreted IgM concentrations by ELISA. Figure 3 shows B < cells that were treated for 30 min at 4°C with a polyclonal rabbit anti-AsGm-1 antisera (150 mg/ml) followed by a 30 min incubation at 37°c in a complement-containing solution in order to lyse AsGm-l + cells. B < cells with or without anti-AsG -l antibody treatment were cult-ured at 2 x ιo 5 cells/ml in the presence or absence of αS-dex (3 ng/ml), and/or IL-1 (100 U/ml) plus IL-2 (150 U/ml). six days after initiation of culture, SN was harvested for determination of secreted IgM concentrations by ELISA. Figure 4 depicts a pure population of activated NK cells were established by IL-2 stimulation, in vitro, of spleen cells derived from scid mice (see methodologies) . 7 days after initiation of scid spleen cell cultures, cells were harvested for flow cytometrio analysis. The following antibodies were utilized: (l) polyclonal rabbit anti-AsGm-l antisera + FITC-labelled polyclonal goat anti-rabbit IgG, (2) FITC-labelled anti-CD3 mAb (2C11) , (3) FITC-labelled anti-Thy-1 mAb (30H12) , (4) FITC-labelled anti-MAC-1 mAb (Ml/70) , (5) FITC-labelled anti-Ia d mAb (MKD6) , and (6) FITC-labelled anti-Ia mAb (Y3-P; control mAb-i.e. scid mutation established on BALB/c background [Ia d ]). Each flow cytometric tracing represents the fluorescence pattern of 10,000 viable cells. Figure 5 shows B s cells that were cultured at 1.25 x 10 5 cells/ml with α«S-dex (3 ng/ml) and/or IL-1 (100 U/ml) plus IL-2 (150 U/ml) in the absence or presence of 10% sort-purified splenic non-B, non-T cells (lgM"CD3") , 10% sort-pύrified splenic T cells (lgM"CD3 + ) , or 3% in vitro-generated pure NK cells. Six days after initiation of culture, SN was harvested for determination of secreted IgM concentrations by ELISA. Figure 6 shows B' p cells that were cultured at 1.5 x 10 5 cells/ml with αtf-dex (3 ng/ml) , IL-1 (150 U/ml) , plus IL-2 (150 U/ml) in the presence of increasing numbers of in vitro-generated pure NK cells (0-80% of B sp cell numbers) . 7 days after initiation of culture, SN was harvested for determination of secreted IgM concentrations by ELISA. 94 -8- Figure 7 shows B sp cells that were cultured at 1.5 x 10 s cells/ml with α<S-dex (3 ng/ml) , IL-1 (100 U/ml) plus IL-2 (150 U/ml) in the absence or presence of varying concentrations of NK-BMF (10-50% v/v) . Six days after initiation of culture, SN was harvested for determination of secreted IgM concentrations by ELISA. Figure 8 shows B 5p cells that were cultured at 1.25 x 10 5 cells/ml with α$-dex (3 ng/ml) , IL-1 (150 U/ml) plus IL-2 (150 U/ml) in the absence or presence of SN obtained 24 h after activation, with plate-bound anti-CD3 mAb (2C11) , of the following CD4 + Thl clones: (1) RA1 (1:0% SN v/v), (2) RA5 (1.0% SN v/v), (3) RA8 (1.0% SN v/v), (4) RC5 (1.0% SN v/v), and (5) RC9 (2.5% SN v/v) . SN was harvested for determination of secreted IgM concentrations by ELISA. Figure 9 shows B sp cells that were cultured at 1.25 x 10 5 cells/ml with α<f-dex (3 ng/ml) , IL-1 (150 U/ml) , plus IL-2 (150 U/ml) in the absence or presence of varying, final concentrations of SN (0.01-1.0% v/v) from anti-CD3-activated Thl clones (see Fig 8) . SN was harvested for determination of secreted IgM concentrations by ELISA. -9- Figure 10 shows a 24 h SN from anti-CD3-activated RA5 Thl clone (T-BMF) was incubated at 37°C in the presence (RA5-SN-PK) or absence (RA5-SN-Ctrl) of proteinase K. B ϊp cells were cultured at 1.25 x IO 5 cells/ml with α<y-dex (3 ng/ml), IL-1 (150 U/ml) plus IL-2 (150 U/ml) in the absence or presence of either RA5-SN-PK (1.0% v/v) or RA5-SN-Ctrl (1.0% v/v). As an additional control, RA5-SN-PK (1.0% v/v) and RA5-SN-Ctrl (1.0% v/v) were added to B ,p cell cultures activated with α5-dex (3 ng/ml) plus IL-5 (150 U/ml) . SN was harvested for determination of secreted IgM concentrations by ELISA. Figure 11 shows a 24 h SN from the anti-CD3-activated RA5 Thl clone (T-BMF) or SN from unactivated resting RA5 cells (Control SN) was tested in an IL-6-specific sandwich ELISA. Recombinant IL-6 was used to generate a standard curve. Figure 12 shows B ,p cells that were cultured at 1.25 x 10 5 cells/ml with α<5-dex (3 ng/ml), IL-1 (150 U/ml) plus IL-2 (150 U/ml) in the absence or presence of varying concentrations of anti-CD3-activated RA5 SN (0.001-1.0% v/v) obtained at various time points after anti-CD3 activation (0-24h) . SN was harvested for determination of secreted IgM concentrations by ELISA. Figure 13 shows B* > cells that were cultured at 1.25 x 10 s cells/ml with α*-dex (3 ng/ml), ι L -ι (150 U/ml) Plus IL-2 (150 U/ml). T-BMF (1% v /v of RA5 SN) was additionally added to replicate cultures on different days (day 0-6, . SN was harvested from all experimental groups 6 days after initiation of culture for determination of secreted IgM concentrations by ELI S A. V I. DETAILED DESCRIPTION OF THE IN V ENTI ON A biological activity has been defined whose dominate function is to potently induce antibody secretion by activated animal B cells. More precisely, applicants have discovered: 1. A cell-free preparation of a novel actilvve substance whose activity is derived from T cells or N K cells which potently induce antibody secretion by activated animal B cells, said substance having Ig secretion activity which is not mimicked by the addition of cytokine in the absence of said cell-free preparation. 2 - τhe cell-free preparation of Claim 1 wherein the active substance is produced by NK cells. 3. The cell-free preparation of Claim 1 wherein the active substance is resistant to 4 -11- neutralization by monoclonal antibodies against cytokines produced by NK cells activated by IL-2 and has Ig secretion activity which is not mimicked by the addition of NK cell cytokines in the absence of said cell-free preparation. 4. The cell-free preparation of Claim 1 wherein the active substance is a protein whose activity is derived from T cells which potently induce antibody secretion by activated animal B cells, said substance: a. is resistant to neutralization by monoclonal antibodies or receptor antagonists against cytokines produced by activated T cells; b. has Ig secretion activity which is not mimicked by the addition of T cell cytokines in the absence of said cell-free preparation; c. can be produced by activation of the T cell through the T cell receptor signalling pathway which induces cytokine production by T cells; d. has activity which is inhibited by treatment with a proteolytic enzyme; e. has activity which is optimal within 3 to 5 hours after activation of the T cell; f. has detectable activity at dilution levels as low as 1:3000; and g. operates as a late acting factor required to induce proliferated B cells to mature into antibody secreting cells. 5. The cell-free preparation of Claim 4 wherein the active proteinaceous substance is a cytokine. 6. The cell-free preparation of Claim 5 which is produced by T cells. 7. The cell-free preparation of Claim 1 which is free of mycoplasm. 8 . The cell-free preparation of Claim 6 wherein the proteinaceous substance can increase the induction of antibody secretion 10 to 40 fold. 9. The cell-free preparation of C laim 3 wherein the active substance can increase the induction of antibody secretion 10"to 30 fold. 1 0 . The cell-free preparation of Claim 1 wherein the active substance whose Ig secretion activity is not mimicked by the addition of T cell or UK cell cytokines comprising Interleukin- 3 • Interleukin-4; Interleukin-6; Interleukin- lθ ; T N F-α; IF N -y; IFN- 7 and TNF-αr; IFN-α.; iFN- and IF N - 7 ; iF N -α and TNF-α; IFN-α, IFN-γ and TNF-α; GM-C S F; and T G F-31. 11- τhe cell-free preparation of Claim 8 wherein the proteinaceous substance is resistant to neutralization by monoclonal antibodies or receptor antagonists against cytokines comprising anti-lFN-γ; TNFRIg; anti-GM-CSF; anti-IL-3; anti-IL-4; anti-lL- 5 ; anti-IL-6; anti-IL-10; and huCD40Fc. 12- The cell-free preparation of Claim 8 wherein the proteinaceous substance is produced by activation of the T cell through the T cell receptor pathway which induces cytokine production by T cells as shown in Figure 12. 13. The cell-free preparation of Claim 9 wherein the active substance is resistant to neutralization by monoclonal antibodies against cytokines comprising anti-TNF-α; Anti-IFN-γ; anti IFNα/γ; anti-IL-4; anti-IL-5; anti-lL-6; and anti-IL- 10. 14. A method of producing a cell-free preparation containing the active proteinaceous substance of Claim 4, comprising: a. coating the bottom of a flask with anti-CD3 antibody; b. adding T cells into the flask and allow said T cells to remain in the flask for a period of 24 hours; c. harvesting the cell-free supernatant; d. inducing the activation and proliferation of B cells in the cell-free supernatant through the B cell antigen receptor mediated signalling pathway utilizing dextran conjugated anti-IgD antibodies in the presence of Interleukin-1 and Interleukin-2 wherein said B cells are at least 98.5 percent purified through fluorescence-activated cell sorting (FACS) ; e. adding activated T cell supernatant; f. harvesting the B cell culture supernatant 6 days after culture; and g. measuring the concentration of secreted IgM in the B cell cultured supernatant by an enzyme-linked Immunosorbent Assay (ELI S A ) . 15. The product produced in accordance with the method of Claim 14. In sum, we have established a novel in vitro assay which has allowed us to screen for potentially novel factors which induce immunoglobulin (Ig) secretion by murine B lymphocytes. We refer to such factors as B cell maturation factors (BMF) . Utilizing this assay we have identified two such activities which we have shown to be distinct from the well-described and characterized cytokines which are known to play a role in B cell activation and differentiation. The first activity was identified in culture supernatants from pure populations of in vitro-generated IL-2-activated murine natural killer (NK) cells. The second activity was identified in the culture supernatants of a panel of anti-CD3-activated murine CD4 + T lymphocyte clones. While these two activities are functionally similar, they could represent distinct molecules, studies to identify the murine BMF from one of the CD4 + T cell clones are currently underway. A cytokine with the ability to induce high rate Ig secretion, or a selective antagonist of such a molecule, could be of potential value as a therapeutic in a diverse number of clinical settings as well as an in vitro tool to generate antibody-forming cells. VII. Methods Mice. Female DBA/2 mice were obtained from the National Cancer Institute (Frederick, MD) and were used at 7-10 weeks of age. The experiments were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Animal Resources, National Research Council, Department of Health, Education, and Welfare. Publ No. (National Institutes of Health) 78-23. Culture medium. RPMI 1640 (Biofluids, Rockville, MD) supplemented with 10% fetal bovine serum (Gibco Laboratories, Grand Island, N.Y.) , L-glutamine (2 mM) , 2-mercaptoethanol (0.05 mM) , penicillin (50 mg/ml) , streptomycin (50 μg/ml) , and genta icin (50 mg/ml) were used for culturing cells. Rea g ents. α<S-dex was prepared by conjugation of H571 (monoclonal mouse IgG2b (b allotype) anti-mouse IgD (a allotype) to a high molecular weight dextran (2 x 10* M.W.). Approximately 6 HcS'/l were conjugated to each dextran molecule. LPS W, extracted from Escherichia coli 0111:B4, was obtained from Difco Laboratories, Inc. (Detroit, MI) and was used at 2 0 mg/ml in all experiments. Affinity-purified, phycoerythrin-labelled polyclonal goat anti-mouse IgM was obtained from Southern Biotechnology Associates (Birmingham, AL) . FITC-labelled monoclonal hamster Ig G anti-CD3e (2C11) monoclonal antibody (mAb) was obtained from Pharmingen (San Diego, CA) . Polyclonal ra b bit anti-AsGm-l was obtained from Wako (Osaka, Japan ) . Murine recombinant (r)IL-l was a gift from Dr. Stephanie Vogel (USUHS, Bethesda, MD) . Murine rIL- 2 was a gift from-Dr. Maurice Gately (Hoffman LaRoche, Nutley, NJ) . Murine rIL-5 was a gift from Dr. Richard Hodes (NIH, Bethesda, MD) . Preparation and culture of B cen«=;. Enriched populations of B cells were obtained from spleen cells from which T cells were eliminated by treatment with monoclonal rat IgM anti-Thy-1 (H013-4) , rat IgG2b anti-CD4 (GK1.5), and rat IgG2b anti-CD8 (2.43), followed by mouse anti-rat Igk (MAR 18.5) and co plement. Cells were then fractionated into high and low density populations by centrifugation over a Percoll gradient (Pharmacia, Piscataway, NJ) consisting of 70, 65, 60, and 50% Percoll solutions (with densities of 1.086, 1.081, 1.074, and 1.062 g/ml, respectively) . The high density cells were collected from the 70 to 66% interface and were used in all experiments. The average percentage of Ig + cells was 85-90% in the 70 to 66% fraction. B cells obtained in this way were referred to as "B cell-enriched" (B') cells. This cell population is also known to contain small numbers of NK cells, macrophages, mast cells, and cells of the granulocytic series. To remove NK cells from the B c cell preparation we further treated B e cells with polyclonal rabbit anti-AsGm-l antibodies (Waco, Osaka, Japan) plus complement as follows: B e cells (IO 7 cells/ml) were incubated in "cytotoxicity medium" (CM) (RPMI + 2.5 mM Hepes + 0.3% BSA) with anti-AsGm-l antibody (150 mg/ml final concentration) at 4°c for 1 h. Cells were then washed once in cold CM and resuspended in CM containing a l/io dilution of rabbit complement (Pel-Freeze, Brown Deer, Wisconsin) at 37°c for 1 h. Cells were then washed and resuspended in medium for further use. Functional assays were carried out in 96-well flat-bottom Costar plates (Costar Cambridge, MA) . Cultured cells were incubated at 3 7° C in a humidified atmosphere containing 6% C0 2 . Establishment of NK cell cultures. Spleen cells from CB-17 SCID mice, obtained from NCI (Frederick, MD) , were cultured in medium at 1 x lθ 7 /ml in the presence of 500 U/ml of rhIL-2. Spleen cells were first treated with anti-Thy-1, anti-CD4, and anti-CD8 + complement as a precaution against the possible presence of small numbers of T cells resulting from "leakiness" in the SCID mutation. NK cells were maintained by splitting them 1:2 into fresh medium + 500 U/ml of IL-2 every 2-3 days. Cells were used for experiments beginning -7-10 days after establishment of culture. Such cells were monitored by flow cytometry to confirm the absence of CD3 + (T) cells. NK cell cultures were re-established every month, using fresh spleen cells from SCID mice, since longer culture periods were associated with deterioration of the NK cell line. Cells derived from CB-17 mice, like those from DBA/2, express H-2 d MHC class I molecules. Preparation of supernatants (SN) from IL-2 activated, in-vitro-σenerated NK cell cultures fNK-BMF) . NK-BMF was prepared in two basic ways: (1) In vitro-maintained, IL-2-activated NK cells were washed and recultured at !.„ x 10 _ cells/ml ^ ^ ^^ of I - 2 (5 00 0/alJ . 24 h later ceu . free cuiture SN κas harvested, betted, and stored at - 2 Ve until used ( 2 ) X, vitro- aintained, I -2-activated NK cells were "ashed 3x and recuitured at !.o x 10 * cells/lnl ,„ ^ presence of s x 10 . B . cells/nl> ^ ^ ^ ^^^ ^ other exogenous stimuli. 24 h later cel l,fr s ^^ S N was harvested, aUquotted, end stored at - 2 o » c untll used C orporation, Seattle, WA. These T cell clones were maintained by weekly stimulation with antigen, spleen cells ( as a source of antigen-presenting cells [APCsJ ) , and exogenous IL-2. Cytokine-containing SN were obtained from cultures of these CD4 + T cell clones in the following manner: Tissue culture wells were incubated with anti-CD3 mAb (2C11) at 10 mg/ml in PBS for 3h at 37°c and then washed 3x in fresh PBS. T cell clones which were allowed to return to their resting state after stimulation with antigen, APCs, and IL-2 by maintaining them for -7 days in the absence of any further exogenous stimuli were added to anti-CD3-coated plates at lxl0 6 /ml for various times, upon which cell-free SN were obtained and either stored at -20°C or 4°C. In the latter case, SN was used in cellular assays within 1-2 weeks of having been harvested. Cvtofluorometric analysis and cell sorting. Spleen cells were stained for 30 min with FITC-labelled anti-CD3 mAb + PE-labelled anti-IgM antibodies (final concentration of 10 mg/ml each in- the presence of a 5-fold excess of anti-FcγRII mAb to prevent cytophilic antibody binding) at IO 7 cells/ml in cold clear HBSS containing 3% FBS and 50 mg/ml each of penicillin, streptomycin, and gentamicin. Cells were then washed and resuspended in staining buffer at IO 7 cells/ml in preparation for fluorescence analysis and/or cell sorting. For analysis, a FACStar Plus or FACSCAN (y Becton Dickinson, Mountainview, CA ) was used and 15,000 cells were collected using logarithmic amplification. Only viable cells were analyzed on the basis of their characteristic forward and side scatter profiles , cell sorting was similarly carried out on a FA CS tar Plus, as we 11 as on an Epics Elite (Coulter Corp., Hialeah, FL) , and sorted cells were immediately reanalyzed to confirm their staining profile. Only sorting purities of >98% were acceptable for subsequent study. S ort-purified B cells ( mIgM +C D 3 -, were referred to as B* cells. Non-B, non-T cells ( nlgKCDT ) and T cells (mIgM C D 3 ÷, were also collected and macrophages were routinely eliminated, during sorting, on the basis of their characteristic forward and side scatter profile C -uantitation of secre P _l g M. i gM concentrations were measured by ELISA, with Immulon 4 , 96 -well flat-bottomed ELI S A plates (Dynatech La b oratories, A lexandria, V A ) . Briefly, ELISA plates were coated with polyclonal goat anti-mouse i gM antibodies (Southern Biotechnology Associates) , followed *y addition of serial dilutions of samples and standards. Alkaline phosphatase-conjugated polyclonal goat anti-mouse lg M antibodies were then added, upon which a fluorescent product was generated from cleavage of 4-methylumbilliferyl phosphate (Sigma ) by specifically-bound alkaline phosphatase-conjugated antibodies. Fluorescence was measured on a 3M 96 fluσrometer (Mountainview, CA) and fluorescence units were converted to Ig concentrations by extrapolation from standard curves determined in each assay by using purified myeloma IgM of known concentration. IgM measurements showed no significant cross-reactivity or interference from the presence of other isotypes (IgD, IgG3, IgGl, IgG2b, IgG2a, IgE, and IgA) . Ouantitation of IL-6. Flat-bottomed, 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated with 6 mg/ml of anti-mouse IL-6 mAb (MP5-20F3) in 0.1 M carbonate buffer (ph 9.6) for 3h at room temperature, blocked with PBS-1% BSA for lh at room temperature, then extensively washed with PBS-Tween. Supernatant or recombinant.purified lymphokine (Pharmingen) diluted in PBS-1% BSA was incubated on these. plates for lh. Biotinylated anti-IL-6 mAb (MP5-32C11) was used at 1 mg/ml and avidin-conjugated alkaline phosph atase at a dilution of 1/2,000. Preliminary experiments showed this assay to be sensitive to 0.22 ng/ml of rIL-6. Specificity was documented by the absence of binding by 1,000-fold higher concentrations of IL-4, IL-5, and IFN- . VIII. EXAMPLES The following working examples are not intended to limit the invention which is defined by the claims appended below. A. Dextran-coniu ated anti-IσD antibodies (α<?-dex) potently induce proliferation of murine B cells in vitro.-We synthesized a novel construct which potently activates murine B cells through the membrane (m)Ig (antigen receptor)-mediated signal transduction pathway. This construct consisted of covalent linkage of multiple anti-IgD monoclonal antibodies (mAbs) to a high molecular weight dextran backbone. We henceforth refer to this construct as α<S-dex (Ref 1) . The high valency of anti-IgD antibodies linked to dextran resulted in extensive B cell mlg crosslinkage at extremely low concentrations of anti-IgD. This resulted in minimal modulation of mlgD from the B cell surface and hence allowed for continuous B cell signalling. Given the small numbers of antigen-specific B cells in the unimmunized mouse, the advantages of cnS-dex lied in its ability to polyclonally activate the vast majority of mature B cells through the mlg signal transduction pathway. Further, aS-dex mimicked the repeating epitope nature of polysaccharides, such as those found within bacterial cell walls, and could be used as an in vitro model to study immune responses to that class of antigens. S-d&x induced resting murine splenic B cells to proliferate but did not, by itself, stimulate Ig secretion. Compared to unconjugated anti-IgD, α£-dex induced substantially higher maximal levels of proliferation at 1000-fold lower concentrations of anti-IgD (Ref 1) . B. Interleukin tIL) -2 or IL-5 induce I secretion in αg-dex-activated B cell-enriched, T cell-depleted spleen cell cultures-Addition of the cytokines IL-2 or IL-5 to αcS-dex-activated B cell cultures resulted in the secretion of large amounts of IgM (Ref 2) . By contrast, unconjugated anti-IgD was ineffective at stimulating cytokine- ediated Ig secretion. Thus, δ-dex represented the first efficient in vitro system for inducing cytokine-dependent polyclonal Ig secretion through the mlg signal transduction pathway. The population of small, resting B cells utilized in these studies were established by depleting T lymphocytes from spleen cells with a cocktail of anti-T cell antibodies and complement followed by fractionation, according to density, by centrifugation on a discontinuous Percoll gradient. Cells were obtained from the high density Percoll fraction and represented cells in the resting or G 0 state of the cell cycle. This population of small T cell-depleted spleen cells consisted of 85-90% B cells with the remainder of cells consisting mostly of NK cells, macrophages, and granulocytes. We henceforth refer to this B cell-enriched spleen cell population as B c cells. IL-2 stimulated Ig secretion by aS-dex-activated B e cells but not by highly purified B cells. To determine whether IL-2 or IL-5 could act directly on the αtS-dex-activated B cell to induce Ig secretion we obtained a highly purified population of small B cells through the use of a florescence activated electronic cell sorter (FACS) (Fig 1). Thus, small B e cells were stained with phycoerythrin (PE)-labelled anti-IgM antibody which selectively binds to mIgM + B cells, plus FITC-anti-CD3 to identify and eliminate any residual T cells and/or cells binding anti-IgM non-specifically. Sort-purified mIgM + B cells were obtained at >99% purity and are henceforth referred to as B ιp cells. Whereas LPS or α<S-dex plus IL-5 stimulation resulted in a comparable level of Ig secretion by small B e and B , cells, activation with αtS-dex plus IL-2 led to Ig secretion only by B e cells (Table 1) . This suggested that a non-B, non-T cell removed during the FACS • procedure was critical for induction of Ig secretion by B sp cells in response to α$-dex plus IL-2. Bcell populations which were less than 98.5% purity typically contained sufficient non-B, non-T cells to allow for induction of Ig secretion in response to a < 5-dex plus IL-2. Hence FACS was required to obtain sufficiently pure B cells to abrogate the αf-dex plus IL-2-induced Ig secretory response. We demonstrated that the Ig secretory response of small B sp cells in response to aS-dex plus IL-2 could be restored by adding back the splenic non-B, non-T cells which were removed by FACS (Fig 2) . The splenic non-B. non-T cells responsible for induction of Ig secretion in a<?-dex plus IL-2-activated B' p cells are AsGm-l*-Depletion of macrophages from the B e cell population, by exploiting the property of macrophages to selectively adhere to plastic, had no effect on Ig secretion in a<S-dex plus IL-2 activated B e cells (data not shown) . Thus, we postulated that the non-B, non-T cell necessary for induction of Ig secretion in this system was an NK cell. Since the vast majority of splenic NK cells, as well as some macrophages, selectively express the marker Asialo Gm-1 (AsGm-1) on their surface, we eliminated NK cells by incubating small B e cells with anti-AsGm-l antibody plus complement. This procedure abrogated the Ig secretory response of B e cells to a«S-dex plus IL-2 strongly suggesting that NK cells were responsible for induction of Ig secretion in this system (Fig 3) . Additional experiments demonstrated that depletion of Ascm-i÷ cells from the small B < cell population had no effect on the Ig secretory response to a,-dex plus IL-5 ( data not shown ) , consistent with the ability of these latter stimuli to act directly at the level of the B cell A pure popm. inn of ISfci.mfifl j g^^ established a pure population of NK cells i n vitro. Thus, we isolated spleen cells from mice which were homozygous for the severe combined immunodeficiency ( scid ) mutation. Such mice genetically lack both B and T cells but * contain functional NK cells. Culture of scid spleen cells in relatively high concentrations of IL-2 resulted, within 6 days, in a pure population of activated NK cells as demonstrated by flow cytometric analysis (Fig 4) . Thus, virtually all cells expressed the NK cell marker AsGm-l as well as Thy-i, and lacked markers for T cells ( C D3) , B cells (la, and macrophages (MAC-l ) . The use of scid spleen cells were essential for obtaining this population since high doses of IL-2 would also expand even small numbers of T cells which might contaminate the B e cell population. Addition of in vitro-generated pure NK cells to α < S-dex plus IL-2-activated B*' cells resulted in induction of Ig secretion which was comparable to that observed when sort-purified splenic non-B, non-T cells were added (Fig 5). Sort-purified small naive splenic T cells, by contrast, were ineffective at inducing Ig secretion in this system. NK cells induced optimal Ig secretion when present at 5-10% of the B cell population ( Fig 6 ) . Addition of conditioned medium from these IL-2-activated NK cell cultures also induced Ig secretion in α.5-dex plus IL-2-activated B ,p cells in the absence of NK cells indicating that NK cells released a BMF (Table 2) . We henceforth refer to this B M F as NK-BMF. Optimal Ig induction by NK-BMF occured at 50 % final volume of NK-BMF with little if any activity observed at- 10% (Fig 7). NK-BMF is not amon g the cytokines known to in d uce B cell activation and maturation. To better characterize the nature of NK-BMF we performed two types of experiments to determine whether NK-BMF was among the cytokines known to induce B cell activation and differentiation. In the first set of experiments we added known cytokines, either singly or in combination, at various concentrations, to cultures of B' p cells stimulated with aS-dex plus IL-2. Neither IL- 3 , i L -4, IL-10, TNF-a, IF N - 7 , lFN-a/0, GM -C S F, or T G F- was capable of inducing Ig secretion in this system. In the second set of experiments we demonstrated that neutralizing antibodies to IL-4, IL-5, i L -lo, TNF-a, IFN- 7 , and IFN-a/0 failed to abrogate the Ig secretary response ( Table 3 ) . In separate experiments, using various concentrations of recombinant IL-12 and a polyclonal goat anti-mouse IL-12 antisera, we also d emonstrated that NK-BMF was not IL-12 ( data not shown ) . As indicated below, several distinct experiments were further carried out which indicated that neither N K-BMF nor T-BMF was IL- 6 ( see Table 4, Table 5, and Figure 11) Anti- C D 3 -activated murine CD4+ T cell clones also release a BMF. Studies with NK-BMF indicated that this activity was present at relatively low titers as indicated by a requirement for its presence at 50% final volume to induce optimal Ig secretion. This has made the further characterization and identification of this activity technically difficult. Since no one has yet been successful at establishing long-term murine NK cell clones we were further hindered by an inability to select for a high BMF-producing NK cell clone. We thus determined whether other cell types were capable of releasing an activity which could similarly induce Ig secretion by a<S-dex plus IL-2-activated B sp cells. Many antigen-specific murine in vitro CD4 + T cell clones have been established in numerous laboratories. It has been well-established that such clones secrete large amounts of cytokines when cultured in tissue culture wells that have been pre-coated with anti-CD3 mAb. CD3 is a component of the T cell antigen-receptor and its crosslinkage induces T cell activation. Further, CD4 + T cell clones have been subdivided into two broad categories dependent upon the profile of cytokines they release upon activation. Thus Thl clones exclusively release IL-2, IFN-γ and lymphotoxin upon activation whereas Th2 clones exclusively release IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. Both types of Th clones share the ability to release TNF-a, IL-3, and GM-CSF. Since we demonstrated that IL-5, present exclusively in SN from anti-CD3-activated Th2 clones, could induce Ig secretion by a<S-dex or aδ-dex plus IL-2-activated B" cells we evaluated Thl clones for their potential release of a novel BMF. Thus, SN from a single anti-CD3-activated Thl clone was added to cultures of α<S-dex plus IL-2-activated B lp cells and was shown to stimulate large amounts of secreted Ig (Table 2) . We similarly tested 5 additional distinct Thl clones for their ability to release a BMF upon anti-CD3 activation. All Thl clones were found to release BMF (Fig 8) . This BMF is henceforth referred to as T-BMF. In contrast to NK-BMF which was required at a 25-50% final volume to induce Ig secretion, T-BMF induced near-optimal Ig secretion at a final volume of 0.1% and continued to have Ig-inducing activity at 0.01% (Fig 9). Thus, BMF-containing T cell supernatants appeared to be at least 250-fold more potent in Ig-inducing activity than supernatants derived from activated NK cell cultures. T-BMF, in the absence of a£-dex+IL-l+IL-2, had no Ig-inducing effect on small B' p cells (data not shown) . T-BMF is a protein and is not among the cytokines known to induce B cell activation and differentiation. That T-BMF is a protein was demonstrated by the ability of proteinase K treatment to abrogate the Ig-inducing activity of T-BMF (Fig 10) . This effect was not due to an inhibitory effect of residual, active proteinase K upon addition of proteinase K-treated T-BMF to the activated B* p cell cultures since addition of proteinase K-treated T-BMF to δ-dex plus IL-5-activated B* cultures did not reduce Ig secretion. To determine whether T-B n MF r wa a s s a a-mn n on nrt g < t-h*.e cy . t_ok,i■nes known to stimulate B cell activation and/or differentiation, we stimulated B* cells with a-5-dex, IL-1, IL-2, and 0.1% T-BMF and added neutralizing antibodies or antagonists to i L - 3 , ι L -4, ι L - 5/ IL _ 6/ IL-10, GM-CSF, TNF-a, and IFN- 7 . none of the antibodies tested inhibited Ig secretion ( Table 4 ) . Since IL-6 has ' been shown to be a cytokine capable of inducing B cell maturation to ig secretion, applicants further tested whether T-BMF was IL-6. T-BMF was not IL-6 as indicated in two distinct ways: ( i) direct measurement of IL-6 in T-BMF, utilizing a highly sensitive ELISA, indicated undetectable amounts of IL- 6 (<200 pg/ml) in undiluted T-BMF (Fig H) . since T _ BMF induces Ig at 0.01% final volume, i L -6 if present would be <0.0 2 pg/ml. Since IL-6 has been reported to exhibit B cell maturation effects only at concentrations in the ng/ml range, it is highly unlikely that T-BMF was IL- 6 . ( 2) Addition of IL-6 from 2-20,000 pg/ml to cultures of α--dex plus IL-2-activated B* cells failed to induce Ig secretion (Table 5) . Finally, since TNF-a and lymphotoxin bind to the same receptor and exhibit nearly overlapping functional effects, it is highly unlikely that T-BMF is lymphotoxin, since TNF-a is not active as a BMF in this system. of a Thl clone-Kinetic studies were performed to determine the time during which T-BMF was induced upon activation with anti-CD3 mAb. Approximately 90% of total T-BMF activity was induced between 2-3 h after anti- C D 3 activation (Fig 12). m the absence of anti-CD 3 no detectable BMF was induced in the Thl clone, T-BMF acts late in culture to stimulate I g secretion by aδ-dex p lus IL-l+IL-2-activate d B cells. To determine the time in culture when T-BMF was required to induce Ig secretion by B cells activated with aδ-dex plus IL-l+IL-2, we established replicate cultures and added T-BMD on different days after initiation of culture. Culture SN was harvested from all experimental groups 6 days after culture initiation. T-BMF strongly induced IG secretion, even when it was added as late as day 3 of the 6 day culture and the level of T-BMF-induced Ig secretion was comparable to that observed when T-BMF was added at initiation of culture (Fig 13) . Kinetic studies in our lab have indicated that the majority of Ig secretion in this system occurs between days 3 to 5 (data not shown) . Addition of T-BMF 1 day after culture initiation induced a substantially higher Ig secretory response relative to cultures in which T-BMF was added at culture initation (Fig 13 ) . Peak Ig secretion, however, occured when T-BMF was added at day 2. Thus, T-BMF acts late in culture to induce Ig secretion, supporting the view that it functions as a true B cell maturation factor. The observation that T-BMF induced opitmal Ig secretion when its addition to culture was delayed by 2 days suggests that either T-BMF or a separate component in the T cell SN in inhibitory for ig secretion when present early in culture, or that T- BMF activity progressively declines during the culture period and is not available in optimal amounts, when added at initiation of culture, for inducing ig secretion late in culture. Table 1 . B c and B sp cells were cultured at 1.25 X IO 5 cells/ml in the presence of a£-dex (3 ng/ml ) , IL-2 (1 5 0 U/ml ) , IL- 5 ( 1 50 U/ml) and/or LPS (20 μg/ml ) . Six days after initiation of culture, SN was harvested for d etermination of secreted IgM concentrations by ELI SA . 5/13294 -37- 1 Table 2 2 IgM secretion (ng/ml) 3 B e B sp 4 Med 26 < 5 6 IL-l/IL-2 54 < 5 7 a«5-dex + 8 IL-l/IL-2 7,500 220 9 aδ-dex + IL-5 23,100 15,000 ' lO NK-BMF 16 12 IL-l/IL-2 + NK-BMF 8,750 13 T-BMF < 5 14 aδ-dex + T-BMF 150 15 a<5-dex + IL-1/IL-2+T-BMF 4,500 16 Table 2. B e and B sp cells were cultured at 1.25 X IO 5 17 cells/ml in the presence of β<J-dex (3 ng/ml) , IL-1 (150 18 U/ml), IL-2 (150 U/ml), IL-5 (150 U/ml), NK-BMF (25% 19 v/v) , and/or T-BMF (25% v/v) . Six days after initiation 20 of culture, SN was harvested for determination of 21 secreted IgM concentrations by ELISA. Table 3 August 1992 IgM secretion (ng/ml) B e cells Medium < 125 aδ-dex+IL-2 13,700 a<S-dex+IL-2+anti-TNF-a 11,500 a6-dex+IL-2+anti-IFN7 15,000 a<S-dex+IL-2+anti-IFNa/3 10,250 a«5-dex+IL-2+anti-IL-4 11,000 a<S-dex+IL-2+anti-IL-5 12,500 a<S-dex+IL-2+anti-IL-6 11,250 aδ-dex+IL-2+anti-IL-lO 13,100 September 1992 B ,p cells IgM secretion (ng/ml) Medium < 35 a.S-dex+IL-2 1,250 α«S-dex+IL-2+NK-BMF 35,000 α<S-dex+IL-2+IL-3 2,000 α<S-dex+IL-2+IL-4 975 aJ-dex+IL-2+IL-10 800 aδ-dex+IL-2+TNF-a 1,250 αδ-dex+IL-2+IFN-γ 2,000 α<J-dex+IL-2+IFN7+TNFa 1,250 a<S-dex+IL-2+IFNa 1 , 875 aδ-dex+IL-2+IFNa+IFN7 1 , 250 β(S-dex+IL-2+IFNa+TNFa 1 , 375 a<S-dex+IL-2+IFNa+IFN7+TNFa 1 , 000 e«S-dex+IL-2+GM-CSF 1 , 313 a<S-dex+IL-2+TGF/Sl 1 , 125 Table 3. (August 1992) . B e cells were cultured at 1.0 x IO 5 cells/ml with a<S-deκ (3 ng/ml), IL-1 (100 U/ml and IL-2 (150 U/ml) in the absence or presence of the following antibodies: (1) anti-TNF-α mAb (XT2 2 ; 50 g/ml) , (2) anti-IFN7 mAb (XMG-6; 50 μg/ml), ( 3 ) polyclonal rabbit anti-IFNa/B anti-serum (1/50 v/v) , (4) anti-IL-4 mAb (llbll; 50 μg/ml), (5) anti-IL-5 mAb (TRFK-5; 50 μg/ml), (6) anti-IL-6 mAb (P7; 50 μg/ml), and (7) anti-IL-10 mAb (SXC; 20 μg/ml) . Six days after initiation of culture, SN was harvested for determination of secreted IgM concentrations by ELISA. (September 1992) . B sp cells were cultured at 1.5 x 10 5 cells/ml with atf-dex (3 ng/ml), IL-1 (100 U/ml) and IL- 2 (150 U/ml) in the absence or presence of the. following cytokines: (1) IL-3 (WEHI 3 SN 25% v/v), (2) rIL-4 (1000 U/ml), (3) rIL-10 (10 U/ml), 4) rTNFa (100 U/ml), (5) rIFNα (10 U/ml), (6) rIFNa (100 U/ml), (7) rGM-CSF (100 U/ml) , and/or (8) purified natural TGF-01 (1.0 ng/ml). Six days after initiation of culture, SN was harvested for determination of secreted IgM concentrations by ELISA. (IL=interleukin, TNF=tumor necrosis factor, IFN=interferon, GM-CSF=granulocyte- macrophage colony stimulating factor, TGF=transforming growth factor) TABLE 4 A. IgM secretion (ng/π aδ-dex + IL-1 + IL-2 82 a«S-dex + IL-1 + IL-2 + RA5-SN 4,000 a<S-dex + IL-1 + IL-2 + RA5-SN + antiIFN-7 3, 250 + TNFRIg 3,620 + anti-GM-CSF 2,400 + anti-IL-3 2,300 + anti-IL-4 4,750 + anti-IL-5 4,000 + anti-IL-10 2,750 + huCD40Fc 4,000 B. aδ-dex + IL-1 + IL-2 3 50 aδ-dex + IL-1 + IL-2 + RA5-SN 15,000 aδ-dex + IL-1 + IL-2 + RA5-SN + anti-IL-6 16,200 Table 4. B' p cells were cultured at 1.5 x IO 5 cells/ml with aδ-dex (3 ng/ml), IL-1 (150 U/ml), IL-2 (150 U/ml) and/or T-BMF (0.1% v/v [24 hr SN from anti-CD3- activated RA5 Thl clone]) in the absence or presence of the following antibodies/antagonists: [A] (1) anti-IFN- 7 mAb (XMG-6; 10 μg/ml), (2) TNFRIg (10 μg/ml; [TNFRIg=tumor necrosis factor receptor-Ig hybrid molecule which binds and neutralizes free TNF-a]), (3) anti-GM-CSF mAb (10 μg/ml/ purchased from Genzyme) , (4) anti-IL-3 mAb (8F8.1, 10 μg/ml), (5) anti-IL-4 mAb (11B11; 10 μg/ml), (6) anti-IL-5 mAb (TRFK-5; 10 g/ml), (7) anti-IL-10 mAb (2A5; 10 μg/ml), (8) huCD40Fc (10 μg/ml; [human recombinant CD40-lg hybrid with binds and neutralizes free CD40 ligand) . [B] anti-IL-6 mAG (MP 520 Pc; 10 μg/ml) . Six days after initiation of culture, SN was harvested for determination of secreted IgM concentrations by ELISA. TABLE 5 IgM secretion (ng/ml) Medium < 100 aδ-dex < 100 aδ-dex+IL-l+IL-2 450 aδ-dex+IL-l+IL-2+T-BMF 4000 aδ-dex+IL-l+IL-2+IL-6 lU/ l 380 10 U/ml 320 100 U/ml 550 1000 U/ml 400 10,000 U/ml 430 Table 5. B" p cells were cultured at 1.5 x 10 5 cells/ml with aδ-dex (3 ng/ml), IL-1 (150 U/ml), in the absence or presence of various concentrations of rIL-6 (1- 10,000 U/ml) and/or T-BMF (1.0% v/v [24 hr SN from anti-CD3-activated RA5 Thl clone]). Sic days after initiation of culture, SN was harvested for determination of secreted IgM concentrations by ELISA.

Claims: WHAT WE CLAIM IS : 1. A cell-free preparation of a novel active substance whose activity is derived from T cells or NK cells which potently induce antibody secretion by activated animal B cells, said substance having Ig secretion activity which is not mimicked by the addition of cytokine in the absence of said cell-free preparation. 2. The cell-free preparation of Claim 1 wherein the active substance is produced by NK cells. 3. The cell-free preparation of Claim 1 wherein the active substance is resistant to neutralization by monoclonal antibodies against cytokines produced by NK cells activated by IL-2 and has Ig secretion activity which is not mimicked by the addition of NK cell cytokines in the absence of said cell-free preparation. 4. The cell-free preparation of Claim 1 wherein the active substance is a protein whose activity is derived from T cells which potently induce antibody secretion by activated animal B cells, said substance: a. i s res i stant to neutralization by monoclonal antibodies or receptor antagonists against cytokines produced by activated T cells; b. has Ig secretion activity which is not mimicked by the addition of T cell cytokines in the absence of said cell-free preparation; c can be produced by activation of the T cell through the T cell receptor signalling pathway which induces cytokine production by T cells; d. has activity which is inhibited by treatment with a proteolytic enzyme; e - has activity which is optimal within 3 to 5 hours after activation of the T cell; f. has detectable activity at dilution levels as low as 1:3000; and g. operates as a late acting factor required to induce proliferated B cells to mature into antibody secreting cells. The cell-free preparation of Claim 4 wherein the active proteinaceous substance is a cytokine. 6. The cell-free preparation of Claim 5 which is produced by T cells. 7. The cell-free preparation of claim 1 which is free of mycoplasm. 8. The cell-free preparation of Claim 6 wherein the proteinaceous substance can increase the induction of antibody secretion 10 to 40 fold. 9. The cell-free preparation of Claim 3 wherein the active substance can increase the induction of antibody secretion 10 to 30 fold. 10. The cell-free preparation of Claim 1 wherein the active substance whose Ig secretion activity is not mimicked by the addition of T cell or NK cell cytokines comprising Interleukin-3; Interleukin-4; Interleukin-6; Interleukin-10; TNF-a; IFN-7; IFN-7 an d TNF-a; IFN-a; IFN-a and IFN-7; IFN-a and TNF-a; IFN-a, IFN-7 and TNF-a; GM-CSF; and TGF-βl . 11. The cell-free preparation of Claim 8 wherein the proteinaceous substance is resistant to neutralization by monoclonal antibodies or receptor antagonists against cytokines comprising anti-lFN-7; TNFRIg; anti-GM-CSF; anti-IL-3; anti-IL-4; anti-IL-5; anti-IL-6; anti-IL-10; and huCD40Fc. 12. The cell-free preparation of Claim 8 wherein the proteinaceous substance is produced by activation of the T cell through the T cell receptor pathway which induces cytokine production by T cells as shown in Figure 12. 13. The cell-free preparation of Claim 9 wherein the active substance is resistant to neutralization by monoclonal antibodies against cytokines comprising anti-TNF-a; Anti-IFN-7; anti IFNa/7; anti-IL-4; anti-IL-5; anti-IL-6; and anti-IL- 10. 14. A method of producing a cell-free preparation containing the active proteinaceous substance of Claim 4, comprising: a. coating the bottom of a flask with anti-CD3 antibody; b. adding T cells into the flask and allow said T cells to remain in the flask for a period of 24 hours; c. harvesting the cell-free supernatant; d. inducing the activation and proliferation of B cells in the cell-free supernatant through the B cell antigen receptor mediated signalling pathway utilizing dextran conjugated anti-IgD antibodies in the presence of Interleukin-1 and Interleukin-2 wherein said B cells are at least 98.5

Inventors: MOND JAMES J, SNAPPER CLIFFORD M, MOND, JAMES, J., SNAPPER, CLIFFORD, M.
Applicants: US HEALTH, THE GOVERNMENT OF THE UNITED STATES OF AMERICA, RE
Application year: 1994
Main classification: C07K14
IPC codes: A61K35, A61K38, A61K39, A61K45, A61P37, C07K14, C07K16